The current application of CAR-T cells in CSCs

The CSCs on the top of the hierarchy cancer are the basement of the heterogeneity and interfere in many signals to communicate with microenvironment. Targeting CSCs can evade the barriers against solid cancers. Many preclinical and clinical research studies have been employed by designing CAR-T cells to target CSCs in cancer immunotherapy. A study using CAR-NK cells to target OCSCs in vitro which target CD133 on ovarian cancer cell lines and are combined with chemotherapy showed a strong antitumor capability.8 A study showed that anti-AC133/CD133+-specific CAR-T cells targeting glioblastoma stem cells have therapeutic efficacy against GBM both in vitro and in vivo, while CD57 expression on T cells is upregulated which CD57 is not a bona fide CSC marker for GBM.5 EpCAM was targeted by CARs and the data demonstrated that anti-EpCAM-specific CARs had apparently antitumor capabilities in prostate cancer and peritoneal carcinomatosis in vitro and in vivo.4,88 Oncofetal antigen 5T4 is predominately expressed in nasopharyngeal carcinoma stem cell-like cells. CAR-T cells targeting 5T4 have anti-nasopharyngeal carcinoma ability when CARs are combined with cytokine-induced killer cells.6 A CAR-T cocktail immunotherapy has been implied on a 52-year-old female with advanced CCA targeting EGFR and CD133 who had gained an 8.5-month partial response (PR) from anti-EGFR CAR and a 4.5-month PR from anti-CD133 CAR suffering from toxicities in the meanwhile (Table 1).7

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(To view a larger version of Table 1, click here.)

As for CAR adoptive cellular immunotherapy in ovarian cancers, the first-generation CAR design targeting α-folate receptor (FR) was first practiced clinically in patients with ovarian cancer in 2006, although no reduction in tumor burden was seen in any patient.90 There have been many studies on ovarian cancer using CAR, and the targets are various and targets are including MUC16,91,92 FR-α,90MUC1,93 NKG2D ligands,94 and mesothelin95,96 all with some barriers. It is noteworthy that anti-CD133 CAR-NK cells combined with chemotherapy have effect on ovarian cancer cell lines in vitro, which tests the determinacy of CD133 as a surface marker of OCSCs, represents the possibility in CAR therapy targeting OCSCs but also lacks powers of persuasion in vivo, and endows universal and exclusive limitation in CAR application.8

The promise and potential pitfalls of targeting OCSCs for CAR-T design

Targeting CSC markers to design CAR to eradicate tumor in vivo or in vitro, is there any advantage to capitalize it on OCSC markers to design CAR? In this scenario, there are three advantages: 1) Ovarian cancer has the tendency of recurrence after a period of clinical recovery from the primary foci, the biological behavior of which can be explained by “tumor stem cell model”.97 A myriad of research studies and practices in the last decades have identified the existence of OCSC population in ovarian cancer which is at the top of the pyramid of cancer ecosystem. Elimination of OCSCs and therapy targeting OCSCs are necessary to combat ovarian cancer; 2) CSCs can resist conventional therapy and in certain cancers have been observed to be enriched after conventional therapy which results in more dismal prognosis of chemotherapy and radiotherapy.98 Immunotherapy has brought major clinical progressing in antitumor therapy. Adoptive cellular immunotherapy labeled by “targeting cancer” is the optimal method, in that, on the one hand it can capture tumor cells by specific reorganization, and on the other hand, it may avoid unpredictable non-cancer cell toxicities to protect normal tissues; and 3) The CAR-T cell is one of the adoptive cellular immunotherapies which come from the bench to the bedside in a more directive reconstruction pathway guided by clinical need in a shorter experimental period. It is independent of MHC-I presentation which is always downregulated in tumor cells especially in CSCs to evade immune system surveillance. Allogeneic T-cell transfer strategies can enlarge the source of T cells for CAR manufacture,84 which will expand the production scale resulting in generic drugs for different patients. Furthermore, the immunogenicity of CARs should be averted to make CAR tools of choice for targeted cancer immunotherapies.99

So far, only one study in vitro has reported about specifically targeting OCSCs by CAR design.8 The paucity of specific markers to identify OCSCs is still pending urgent resolution. Ovarian cancer is intratumoral heterogeneous and harbors many pathology types which have different origins,100 and there may be more than one group of OCSCs in a tumor; hence, the heterogeneity of OCSCs cannot be identified by the only marker or group of markers, in that, even the most common markers of OCSCs are found in only <40% of tumors.18 In addition, unexpected toxicities emerge even though CAR-T cells have been tested in vitro tests and then were applied in clinical trials including cytokine release syndrome, neurologic toxicity, “on target/off tumor” recognition, and anaphylaxis, which calls for more safe administration undoubtedly.101

Although the CAR-T therapy has been hard-hitting therapy for numerous cancers, considering it as regimen for patients with ovarian cancer still has pitfalls. 1) Immunotherapy must combine other conventional therapies. Primary cytoreductive surgery should be considered as the gold standard to eliminate macroscopic foci for patients with advanced epithelial ovarian cancer.102 Chemotherapy and radiotherapy have decreased mortality rate of ovarian cancer in last decades. Meanwhile, ovarian cancer cytoreduction can induce transformation of T-cell ratio in the tumor which will enhance immune function against cancer.103 Epigenetic inhibitors such as DNA demethylation agents were examined in the clinic against chemotherapy-resistant ovarian cancer.104 Since the management of ovarian cancer needs is a multidisciplinary teamwork,105 “combination” is the headline of individual therapy referring to surgery, chemotherapy, epigenetic drugs, and immunotherapy. 2) OCSC is a rare population of the cancer; CAR designed for CSCs must combine with other immunotherapies such as the blockade of immune checkpoints, antibodies, and innate immunotherapy. Within CAR design, modifying more function parts is under hot investigation. For example, “combinatorial antigen recognition with balanced signaling” is an engineered T cell designed with a CAR and a chimeric costimulatory receptor (CCR). The CAR and CCR can recognize respective antigens which suggests that only when the two antigens are on the tumor cell surface, this CAR-T can kill the tumor cell.106 This way could suit OCSCs well because there are coexpression markers on OCSCs such as CD133+/EpCAM+, and this way will improve the specificity of CAR-T cells by preventing healthy cells from killing which is called “on antigen off target” effect. Different CAR-T cells can be used in the same patient targeting antigens sequentially which is called cocktail therapy, such as the case reported on a female with advanced CCA who was treated with targeting EGFR CAR-T cells and anti-CD133 CAR-T cells and had mitigation when considering the disease progression.7 3) A certain marker or a group of markers specific for CSCs that can be used to identify CSCs as an exact standard has not yet been found. Different markers can be found in the same cancer type which may imply that heterogeneity is not only in a tumor but also exists inside CSCs, so what is the earliest ancestor of CSCs is not certain yet or the current pathology cancer type standard does not suit for CSCs to match the disease. Some surface markers of stem cells are not always carried on the CSCs, which may be replaced by non-CSCs making up the bulk of tumor, so “relying on markers will fool you,” suggesting that the identification of CSC and non-CSC is not consistent indeed.107 It is probably due to that some non-CSCs can dedifferentiate into CSCs with a dynamic transformation. The therapy targeting CSCs should be combined not only mutually in the same treatment course but also in a scheme containing different steps of the tumor progressing in macro and various cell cycles in micro which is rigorously individual for every patient. 4) The most ideal markers on OCSCs as CAR-T antigens have not yet been discovered to our knowledge. For instance, CD133 which is the most widely investigated in CSCs of many cancer types is expressed in normal brain tissues, hematopoietic stem cells, and endothelial progenitor cells.24 Anti-CD133 CAR-T may result in toxicity for normal tissues (“on antigen off target”) without combined balance modification in CAR design to avoid side effects. More safe and effective strategies about CAR-T cells and immunotherapy by combination with other conventional therapy to eliminate cancer need further research.